Procedures for Analysis of Dried Plasma Using Microsampling Devices to Detect Sulfur Mustard-Albumin Adducts for Verification of Poisoning

Abstract

Incorporation of the chemical warfare agent sulfur mustard (SM) produces a covalent adduct with human serum albumin (HSA) representing an established plasma biomarker of poisoning. Bioanalytical verification requires both plasma generation from whole blood and shipping to specialized laboratories following strict guidelines for complex packaging. These needs often push the infrastructural boundary in crisis regions and war zones. Therefore, we herein originally introduce different reliable bioanalytical procedures using filter paper as well as novel volumetric microsampling tools (Mitra devices and Noviplex DUO cards) to generate dried plasma samples not liable to the shipping constraints. In addition, the Noviplex device enables in-transit separation of plasma from whole blood without the need of a centrifuge. Plasma-loaded and dried devices were subjected to pronase treatment yielding the alkylated dipeptide hydroxyethylthioethyl-CysPro (HETE-CP) derived from the HSA-SM adduct that was detected by microbore liquid chromatography-electrospray ionization tandem-mass spectrometry (μLC-ESI MS/MS). For all devices, samples exposed to SM yielded excellent linearity (0.025-50 μM SM) and good precision (≤13%) and fulfilled forensic quality criteria for ion ratios of qualifying and quantifying product ions. Stability of the HSA-SM adduct in dried and liquid plasma is shown under conditions of three climatic zones (temperate climate, hot and dry climate, and hot and humid climate) for at least 9 days simulating the period of delayed sample shipping. Our results originally document that dried plasma is appropriate for storage and shipping at ambient temperature and that novel microsampling tools are of essential benefit when targeting the HSA-SM adduct for verification analysis.

Methionine329 in human serum albumin: A novel target for alkylation by sulfur mustard

“…Therefore, toxicity of SM is assumed to be due to the alkylation of proteins and DNA resulting in the functional impairment of cells, tissues, organs, and physiological processes. 12,[17][18][19][20][21][22][23] Based on the proteolysis of alkylated HSA, several analytical methods targeting the Cys 34 modification as HETE-CysProPhe (HETE-CPF) and HETE-CysPro (HETE-CP) were developed and have proven to be reliable biomarkers of exposure in vitro, in vivo, and in real cases of SM poisoning. 2 For the verification of an alleged use of SM, protein adducts reveal beneficial long-term stability of several weeks or even months in vivo when compared to products of SM hydrolysis and enzymatic biotransformation.…”

“…Due to its bifunctional structure, crosslinks between biomolecules may occur. 12,[15][16][17][18][19][20][21][22][23][24] Herein, we present Met 329 as an additional target of SM in HSA. 15,16 Most relevant biomarkers are alkylated peptides derived from human serum albumin (HSA).…”

Identification of novel disulfide adducts between the thiol containing leaving group of the nerve agent VX and cysteine containing tripeptides derived from human serum albumin

“…While phosphylation is the critical step of AChE inhibition by modification of its active site serine residue, we have recently shown that the corresponding leaving group of VX (2-(diisopropylamino)ethanethiol, DPAET) is also capable of forming adducts with endogenous cysteine residues of HSA [25] . Cys-Pro dipeptides including the only free cysteine sidechain at position 34 (C 34 P) were already known to result from pronase cleavage of HSA and facilitated the identification of DPAET-CP as a novel biomarker for VX exposure in vitro [8,[14][15][16][17]25] . Enzymatic cleavage of HSA incubated with VX using pronase resulted in cysteine and proline containing small peptides.…”

“…However, today these compounds are banned by the Chemical Weapons Convention (CWC) comprising strict surveillance by the Organization for the Prohibition of Chemical Weapons (OPCW, Nobel Peace Prize 2013) [3] . For this purpose, reliable analytical methods are required allowing verification of an alleged use of CWA [5][6][7][8] . For this purpose, reliable analytical methods are required allowing verification of an alleged use of CWA [5][6][7][8] .…”

N‐Acetylcysteine as a chemical scavenger for sulfur mustard: New insights by mass spectrometry

“…In this study, reactions with human serum albumin (HSA) containing the non‐disulfide bridged Cys 34 ‐residue were characterized. HSA is a suitable biomarker for SM exposure by forming HETE‐HSA but will also react with thiol compounds like NAC forming disulfide‐bridged adducts . According to Harada et al, the NAC‐HSA adduct formation happens in 2 steps .…”